B cells have long been regarded as having the primary duties of promoting immunity by presenting antigen to T cells and producing antibody. More recently it has been appreciated that, similar to the T cell arm of the adaptive immune response, the B cell arm is charged with significant regulatory responsibility. This was first appreciated in experimental autoimmunity models in which the absence of B cells exacerbated disease. In the transplant arena, we first demonstrated a model of transplant tolerance (based on anti-CD45RB antibody therapy) that was dependent on the presence of B cells. Since this finding, a number of investigators have substantiated the generality of this property, showing a similar B cell requirement using other tolerogenic regimens including co-stimulation blockade, anti-TIM-1, and anti-TIM-4, and we recently reported the combination of anti-CD45RB and anti-TIM-1 to behave similarly in more stringent strain combinations. These models provide opportunity to delineate the mechanism of action of Bregs, to examine their role in rejection and tolerance, and to begin to explore their potential as a cellular therapeutic. During the last funding period we made significant progress in defining the in vivo action of Bregs. Some of the key findings that set the stage for the current proposal include that tolerance induced by anti-CD45RB and anti- TIM-1 treatment is B cell dependent and can be adoptively transferred from tolerant hosts to both B cell deficient and immunologically replete untreated mice. We were also the first to report that antibody induced tolerance and adoptive transfer of Breg tolerance is dependent on host Tregs. In addition, we recently determined that in our tolerance model, TGF-? is required for development of tolerance and, specifically, that B cell secretion of TGF-? is essential. This provides a natural link between Bregs and Tregs and will be dissected further in the proposed studies of Aim I. We recently initiate studies of nave B cells activated by TLR ligands that also exhibit graft survival prolonging regulatory properties. In Aim II, compare the mechanism of action of these cells with those recovered from tolerance hosts. Finally, in Aim III, we begin to explore the potential of in vitro expanded Bregs (eBregs) both to facilitate our mechanistic analyses and to begin to assess the translational potential of Bregs as a means to control alloimmunity and autoimmunity.